烟草丛顶病毒RdRp介导的体外复制调控研究

发布时间：2018-04-20 03:25

  本文选题：烟草丛顶病毒 + RNA依赖的RNA聚合酶　； 参考：《山东农业大学》2017年硕士论文

【摘要】：烟草丛顶病毒(Tobacco bushy top virus,TBTV)为番茄丛矮病毒科(Tombusviridae)幽影病毒属(Umbravirus)成员,与黄症病毒科(Luteoviridae)的烟草扭脉病毒(Tobacco vein distorting virus,TVDV)复合侵染引起烟草丛顶病。TBTV基因组是一条(+)ssRNA,由4 152个核苷酸组成,编码4个开放阅读框。ORF1编码35 kDa的蛋白,ORF1的C端与ORF2的N端有8个密码子的重叠,ORF1蛋白通过-1位的移码翻译机制表达产生RdRp,为ORF1/ORF2融合蛋白形式。在对云南多地采集的烟草丛顶病毒进化分析后发现,烟草丛顶病毒RdRp编码区序列一致率相对较低,推测RdRp的活性变化可能导致病毒致病力的变化。本研究旨在建立TBTV RdRp介导的体外复制体系,并初步定位参与复制调控的RNA元件。首先通过重叠PCR扩增得到烟草丛顶病毒中国分离物RdRp的编码序列。构建以pMAL-C2X为基础载体的原核表达载体pMAL-TB-RdRp。0.5 mM IPTG诱导可特异性表达分子量约为120 kDa的MBP-RdRp融合蛋白。不同温度条件下诱导MBP-RdRp,结果显示,相比26℃和37℃,18℃下诱导表达的MBP-RdRp融合蛋白的可溶性比例较高,约17%;经亲和层析纯化的MBP-RdRp可特异性识别TBTV正链和负链的3'末端序列,催化体外复制;并且对正负链的3'末端的体外复制效率存在差别,识别负链3'末端的体外复制效率明显高于正链3'末端。说明TBTV RdRp介导的特异性体外复制的建立。同时,结合RNA结构体外分析构建了3'UTR区的部分突变体,根据体外复制效率的变化初步定位了参与复制调控的RNA元件。同时,构建了不同分离物TB-JC,TB-MDI,TB-MDII的RdRp的原核表达载体,并初步验证了纯化的不同分离物RdRp的体外复制活性。为将来进行不同TBTV分离物RdRp的活性比较并定位关键氨基酸突变奠定了基础。
[Abstract]:Tobacco bushy top virus (TBTV), a member of Tombusviridaevirus, a member of the genus Umbravirus, is infected with tobacco vein distorting virusTV DVV of Luteoviridae.TBTV genome is a single (ssRNAs, composed of 4 152 nucleotides). ORF1, which encodes four open reading frames. ORF1 encodes 35 kDa, is expressed in the form of ORF1/ORF2 fusion protein, with 8 codon overlapped ORF1 proteins at the N end of ORF2 and expressed by the 1-bit frameshift translation mechanism. Based on the evolutionary analysis of tobacco top virus collected in Yunnan province, it was found that the consistent rate of RdRp coding region of tobacco top virus was relatively low. It was speculated that the change of RdRp activity might lead to the change of virulence of the virus. The aim of this study was to establish an in vitro replication system mediated by TBTV RdRp and to locate the RNA elements involved in replication regulation. Firstly, the coding sequence of the Chinese isolate RdRp of tobacco cluster top virus was obtained by overlapping PCR amplification. A prokaryotic expression vector pMAL-TB-RdRp.0.5 mm IPTG based on pMAL-C2X was constructed to induce the specific expression of MBP-RdRp fusion protein with molecular weight of about 120 kDa. The results of induction of MBP-RdRpat at different temperatures showed that the soluble ratio of the MBP-RdRp fusion protein induced at 26 鈩，

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